化学生物学与合成生物学课件

化学生物学与合成生物学化学生物学与合成生物学王江云王江云化学生物学与合成生物学Fossils present but rareEvolution and expansion of lifePlants invade the landAge of reptilesAge of mammalsInsects and amphibians invade the land近近4040亿年的进化历程，创造了丰亿年的进化历程，创造了丰富多彩的生物。据估计地球物种富多彩的生物。据估计地球物种总数多达总数多达1 1亿种。亿种。亚里斯多德亚里斯多德达尔文达尔文沃森克里克沃森克里克人类基因组人类基因组文特文特人类对生命本质人类对生命本质的研究有两千多的研究有两千多年了。基因组测年了。基因组测序之后，我们对序之后，我们对生物的认识更加生物的认识更加深入，而对生命深入，而对生命本质的理解还远本质的理解还远远不够远不够千人基因组千人基因组不能创造的东西就不能被理解。不能创造的东西就不能被理解。人工生命可以更好理解生命本质。人工生命可以更好理解生命本质。人工合成生命的重大科学意义人工合成生命的重大科学意义 Fossils present but rareEvolut改造生命，造福人类快速发展的领域，生命科学的新方法工程、生物和信息科学的多学科综合交叉产生广泛的工业应用 环境健康新医药新材料新能源化工绿色农业人人工工生生命命人工合成生命的重大科学意义人工合成生命的重大科学意义 改造生命，造福人类 环境健康新医药新材料新能源化工绿色农业人化学生物学与合成生物学课件化学生物学与合成生物学课件我我们应该做什么？做什么？化能自养化能自养光光能自养能自养有氧代谢有氧代谢能量利用水平是人类进步的标志电能不能在自然界稳定存在，自然进化生物不能高效利用电能人工合成电能、光能、生物质能电能、光能、生物质能高效利用的新生命体系具有重要意义。风能风能水能水能核能核能太阳能太阳能电电能能新人工生命体系新人工生命体系我们应该做什么？化能自养光能自养有氧代谢能量利用水平是人类进化学生物学与合成生物学课件化学生物学与合成生物学课件化学生物学与合成生物学课件化学生物学与合成生物学课件化学生物学与合成生物学课件二氧化碳还原酶在电极上的定点偶联二氧化碳还原酶在电极上的定点偶联二氧化碳还原酶在电极上的定点偶联利用电能进行淀粉生物合成利用电能进行淀粉生物合成利用电能进行淀粉生物合成形成电能细胞，创新生物的能量来源，引领下一代生物技形成电能细胞，创新生物的能量来源，引领下一代生物技术的发术的发展展，如生物计算机、生物传感器、分子马达等。，如生物计算机、生物传感器、分子马达等。电子催化酶与电子传递通道设计电子催化酶与电子传递通道设计电能驱动电能驱动人工生命体系人工生命体系电能驱动电能驱动线粒体基因组人工合成与优化组装线粒体基因组人工合成与优化组装电能高效利用电能高效利用形成电能细胞，创新生物的能量来源，引领下一代生物技术的发展，Metalloenzymes play important roles in Metalloenzymes play important roles in Metalloenzymes play important roles in aa alternative lternative lternative ee energiesnergiesnergiesMost important enzymes for sustainable energy are metalloenzymes.(cytochrome c oxidasehydrogenase)(photosystem I)(photosystem II)(lignin peroxidaseManganese peroxidase)Fuel cellsHowever,metalloenzymes are too expensive.Fossil fuels?Metalloenzymes play important Why designing artificial biocatalysts?Why designing artificial biocatalysts?Approaches to novel biocatalystsApproaches to novel biocatalysts:Top down:reprogramming native enzymes Top down:reprogramming native enzymes Bottom up:design and engineering artificial biocatalystsBottom up:design and engineering artificial biocatalystsWhy designing artificial bioca16Why designing artificial enzymes?Why designing artificial enzymes?nUltimate test our knowledgenReveal new conceptsnDesign new biocatalysts with unprecedented new propertiesnProvide cheaper alternatives for biotechnological and pharmaceutical applications Engineering artificial biocatalystsY.Lu,et al.Nature 460,855-862(2009).Approaches to novel biocatalysts:Top down:reprogramming native enzymes Bottom up:design and engineering artificial biocatalystsWhy designing artificial enzym17Challenges and opportunities in Challenges and opportunities in designing metalloenzymes as artificial biocatalystsdesigning metalloenzymes as artificial biocatalysts A wide number of metal ions and difference oxidation states of the same metal ions;Structural features(bond distance,angle and geometry)vary widely and ill-defined;Most metal ions have beautiful colors and strong magnetic properties,serving as in situ probe of the design process.J.B.,Siegel et al.Science 329,309-13(2010).Y.Lu,et al.Nature 460,855-862(2009).Challenges and opportunities i18Advantages of Biocatalysts in fuel cellsAdvantages of Biocatalysts in fuel cellsAdvantages of Biocatalysts in fuel cellsCytochrome c oxidase(CcO)is the best fuel cell catalysts:Has much lower overpotential(190-370 mV)uses earth abundant metal ions(iron and copper)C.H.Kjaergaard,J.Rossmeisl,and J.K.Nrskov,Inorg.Chem.49,35673572(2010).Advantages of Biocatalysts in His291His290His240Tyr244CuB Bovine heart CcO(PDB ID:1OCC):membrane protein 13 subunits,MW=200 kDa(1850 amino acids)T.Tsukihara,et al.,Science 269,1069-1074(1995)S.Iwata et.al.,Nature 376,660-669(1995)Cytochrome c Cytochrome c Oxidase(CcO)Oxidase(CcO)Heme-CuB centerCuA centerCcO is a large membrane protein and unstable for fuel cell applications.His291His290His240Tyr244CuB BoUsing a small protein azurin to model CuUsing a small protein azurin to model CuA A in CcO in CcOIwata,S.;Ostermeier,C.;Ludwig,B.;Michel,H.Nature 376,660-669(1995).Hay,M.T.;Richard,J.H.;and Lu,Y.Proc.Natl.Acad.Sci.U.S.A.93,461-464(1996).The designed CuA looks almost identical to the native CuA.B.CuA in Azurin(P.aeruginosa)(designed CuA,1.65)A.CuA in CcO(P.denitrificans)(native CuA,2.7)Y.Lu,et al.Nature 460,855-862(2009).Using a small protein azurin t21Overcoming over-potential problem:rationally tuning Overcoming over-potential problem:rationally tuning redox potentials without affecting the active siteredox potentials without affecting the active site+140 mV+140 mV 110 mVNicholas M.Marshall,Dewain K.Garner,Tiffany D.Wilson,Yi-Gui Gao,Howard Robinson,Mark J.Nilges,Yi Lu Nature 462,113-116(2009).Native azurinY.Lu,et al.Nature 460,855-862(2009).Overcoming over-potential prob22How good is it?How good is it?Sohini Mukherjee,Sabyasachi Bandyopadhyay,Arnab Mukherjee,Yi Lu,Abhishek DeyHow good is it?Sohini MukherjeHow good is it?How good is it?Sohini Mukherjee,Sabyasachi Bandyopadhyay,Arnab Mukherjee,Yi Lu,Abhishek DeyCatalystkcatTurnover No.Native CcO105 M-1 s-1-Synthetic model1.2 x 105 M-1 s-11.2 x 104Designed protein4.6 x107 M-1s-11.0 x 105J.P.Collman,et al.Science 315,1565(2007).How good is it?Sohini MukherjeIntroducing unnatural amino acid into Introducing unnatural amino acid into artificial biocatalysts artificial biocatalysts Xiaohong Liu,Yang Yu,Cheng Hu,Wei Zhang,Yi Lu,Jiangyun Wang Angew.Chem.,Int.Ed.51,4312-4316(2012).The cross-linked His-Tyr increased the reactivity and turnovers dramaticallyKcat=120 S-1Introducing unnatural amino ac25Lignin biodegradationLignin is the second most abundant biopolymer on earth next to celluloseA critical barrier to biomass conversionWhile rot fungi is known degrade lignin naturally,but is very difficult forlarge-scale industrial applications White rot fungiLignin biodegradationLignin is26Bakers yeast vs.white rot fungi Bakers yeastBakers yeastWhite White rot fungi rot fungi Bakers yeast is much cheaper and widely available for industrial applicationsBakers yeast vs.white rot fu27Cytochrome c peroxidase (CcP)Cytochrome c peroxidase (CcP)Manganese peroxidase(MnP)Manganese peroxidase(MnP)CcPCcP MnPMnP(bakers yeast)(bakers yeast)(white rot fungi)(white rot fungi)Oxidation of cyt Oxidation of cyt c c Degradation of lignin and many Degradation of lignin and many aromatic pollutants including PCBs aromatic pollutants including PCBs1.B.C.Finzel,T.L.Poulos,and J.Kraut,J.Biol.Chem.259,13027(1984).2.M.Sundaramoorthy,K.Kishi,M.H.Gold,and T.L.Poulos,J.Biol.Chem.269,32759(1994).Cytochrome c peroxidase (CcP)28Active Site CcP(Bakers yeast)MnP(white rot fungi)CcP and MnP are very similar.Active SiteCcPMnPCcP and MnP a29Major Difference Trp Trp 5151Trp 191Trp 191Mn(II)Mn(II)Phe 45Phe 45Phe 190Phe 190B.KS Yeung,X.Wang,J.A.Sigman,P.A.Petillo,and Y.Lu Chem.&Biol.4,215(1996).X.Wang and Y.Lu,Biochemistry,38,9146-9157(1999).A.Gengenbach,S.Syn,X.Wang,and Y.Lu,Biochemistry,38,11425-11432(1999).CcP(Bakers yeast)MnP(white rot fungi)Major DifferenceTrp 51Trp 191M30Transforming CcP into functional MnPTransforming CcP into functional MnPMnCcPW191FMnCcPMnCcPW191F/W51FMnCcPW51FWTCcPNo enzymeB.KS Yeung,X.Wang,J.A.Sigman,P.A.Petillo,and Y.Lu Chem.&Biol.4,215(1996).X.Wang and Y.Lu,Biochemistry,38,9146-9157(1999).A.Gengenbach,S.Syn,X.Wang,and Y.Lu,Biochemistry,38,11425-11432(1999).Transforming CcP into functionImproving KImproving KMM through engineering non-through engineering non-covalent interactionscovalent interactionsY36FK179RI40GH-bondingMobility of ligandSteric clashesFlexibilitySalt bridgeH-bondingMnCcPImproving KM through engineeriActivity assays of triplet mutant on lignin Activity assays of triplet mutant on lignin modelsmodelsn nTested activity assays on:Tested activity assays on:Dimeric lignin model Dimeric lignin model compound(HPLC analysis)compound(HPLC analysis)n nGuaiacylglycerol-guaiacol Guaiacylglycerol-guaiacol ether*ether*Alkali-treated lignin Alkali-treated lignin(HPLC)(HPLC)*LigninmodeliscourtesyofDr.Michelle Chang fromUCBerkeley.See:BrownMEetal,ACSChem.Biol.2012,7,2074-2081Activity assays of triplet mutFluorescent protein design for Mn(III)sensing Liu,X.;Wang,J.*J.Am.Chem.Soc.2014,136,1309413097.Fluorescent protein design for34Liu,X.;Wang,J.*J.Am.Chem.Soc.2014,136,1309413097.Fluorescent protein design for Mn(III)sensing Liu,X.;Wang,J.*J.Am.C35CO2+H2O (CH2O)n+O22H2OhvO2+4H+4e-NAPHCO2+H2O (CH2O)n+O22H2OhvO237Green fluorescent protein:Efficient photo trigged electron transferLV,X.;Wang,J.*Angew.Chem.Intl.Ed.201437Green fluorescent protein:L化能自养化能自养光光能自养能自养有氧代谢有氧代谢能量利用水平是人类进步的标志电能不能在自然界稳定存在，自然进化生物不能高效利用电能人工合成电能电能、光能、生物质能光能、生物质能高效利用的新生命体系具有重要意义。风能风能水能水能核能核能太阳能太阳能电电能能新人工生命体系新人工生命体系化能自养光能自养有氧代谢能量利用水平是人类进步的标志风能水能